Influenza viruses are classified as A, B, and C genera within the family Orthomyxoviridae, which are enveloped, negative-sense (complementary to mRNA sequence), single-stranded RNA viruses with a segmented genome. Influenza A and B type viruses, which possess eight gene segments, evolutionarily diverged from each other more recently than influenza C viruses which only have seven discrete gene segments (Suzuki and Nei, 2002). Influenza A viruses (IAV) infect a variety of warm-blooded animals including humans, horses, pigs, etc., and aquatic birds serve as their natural reservoir (Webster et al., 1992).
Compared to IAV, influenza B and C viruses which mainly infect humans are less common and usually cause mild illness (Taubenberger and Morens, 2008). IAV can be further grouped into different subtypes based on the antigenicity of the two major surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). To date, there are 16 recognized HA subtypes and nine NA subtypes (Fouchier et al., 2005), and recently a new IAV has been discovered with both HA and NA which are divergent from all known influenza A subtypes (Tong et al., 2012). Each influenza virus strain is designated according to its type, the host of origin (if non-human), site of isolation, isolate number, year of isolation, and in the case of IAV, the subtype of HA and NA is given in parentheses (W.H.O., 1980). For example, A/Uruguay/716/2007 (H3N2) is the 716th isolate of a H3N2 subtype IAV isolated from a person in Uruguay in 2007.
The genome of IAV consists of eight RNA segments that typically encode a total of eleven proteins (Ghedin et al., 2005): polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), PB1-F2, polymerase acidic protein (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), the matrix protein (M1), the ion channel protein (M2), nonstructural protein 1 (NS1) and nuclear export protein/nonstructural protein 2 (NEP/NS2).
Among the eleven proteins, M2 and NEP/NS2 are encoded by spliced mRNAs from M and NS gene segments, respectively (Lamb and Horvath, 1991). PB1-F2 has been found to be encoded by an alternate open reading frame near the 5′ end of the PB1 gene in most IAV (Chen et al., 2001). The PB1 gene recently has been reported to encode a third polypeptide expressed via differential AUG codon usage, termed N40 (Wise et al., 2009). Each viral segment contains noncoding regions at both 5′ and 3′ ends with promoter activity; the first 12 nucleotides at the 3′ end and the last 13 at the 5′ end are highly conserved among all segments, and these are followed by segment-specific noncoding regions (Fodor et al., 1995; Parvin et al., 1989). These conserved regions are also found to incorporate the RNA packaging signals for virus assembly (Gog et al., 2007).
The IAV particles are pleomorphic with spherical or filamentous morphology, or a mixture of both. Fresh clinical isolates are mostly seen as filamentous particles in contrast to the laboratory strains which have been extensively passaged in eggs or tissue culture are more in spherical shape (80-120 nm in diameter). The lipid envelope of the influenza virus particle is derived from the host cytoplasmic membrane embedded with two major integral membrane glycoproteins or spikes, HA and NA, projecting from the surface. The mean ratio of HA to NA spikes is about 4:1 and both protrude from the viral surface ranging from 10-12 nm in length (Nayak et al., 2009). The HA molecules are rod-shaped while NA spikes resemble ‘mushroom’ with a hydrophobic stalk. Indirect immunogold staining showed that the HA spikes are uniformly distributed on the virions (Murti and Webster, 1986) while the distribution of NA remains uncertain. It has been shown that if the HA spikes are removed with trypsin, then NA spikes became evenly distributed (Erickson and Kilbourne, 1980). However, earlier observations suggested that the NA spikes are clustered in discrete areas (Compans et al., 1969) as shown by immunoelectron microscopy with monoclonal antibodies, the NA proteins seem to be in patches (Amano et al., 1992; Murti and Webster, 1986). The third transmembrane protein, M2, serves as an ion channel to pump protons into the virion core during the uncoating process which releases the viral genome (Pinto et al., 1992; Sugrue et al., 1990).
Beneath the lipid membrane, the matrix protein M1 functions as a bridge between the envelope and the central virion core composed of eight ribonucleoprotein complexes (RNPs) (Nayak et al., 2004; Schmitt and Lamb, 2005). The M1 layer in opposition to the lipid membrane is believed to stabilize the virus particle (El Karadaghi et al., 1984).
Immunogold labeling with monoclonal antibodies to M1 failed to detect the protein in virions unless they were first treated with a protease or a detergent (Murti et al., 1992). It has been demonstrated that M1 directly binds lipid membrane (Bucher et al., 1980; Ruigrok et al., 2000) and associates with the transmembrane proteins: HA, NA, and M2 (Ali et al., 2000; Enami and Enami, 1996). M1 was also shown to interact with viral RNPs (vRNPs) and the M1-vRNP complex can be isolated from either infected cells or purified virions (Hara et al., 2003; Kawakami and Ishihama, 1983). The isolated vRNPs are rod-shaped, right-handed helices in various lengths ranging from 50 to 150 nm (Compans et al., 1972). Each RNP is comprised of one set of polymerase complex (PB1, PB2, and PA) and one viral RNA segment coated by NP with approximately one NP per 25 nucleotides and without sequence specificity (Compans et al., 1972; Ortega et al., 2000). The partially complementary 5′ and 3′ terminal ends of viral RNA form a panhandle-like structure (Hsu et al., 1987). NEP/NS2, an exporter for RNP complexes from the nucleus, is also found in purified virions, whereas NS1 and PB1-F2 proteins have not been detected (Richardson and Akkina, 1991).
Hemagglutinin (HA) and neuraminidase (NA) are the two major viral surface glycoproteins and the most important immunogens recognized by the host adaptive immune system. Accordingly, IAV can be further divided into 16 different HA subtypes (H1-H16) and 9 different NA subtypes (N1-N9) based on the differential antigenicity of HA and NA molecules (Fouchier et al., 2005). Phylogenetically, there are two groups of HAs: group 1 including H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16 and, group 2 which contains H3, H4, H7, H10, H14, and H15. Similarly for NA, group 1 includes N1, N4, N5, and N8, and group 2 includes N2, N3, N6, N7 and N9 (FIG. 2).
The HA protein is a rod-shaped homotrimer with the carboxyl terminus integrated into the viral lipid membrane and the hydrophilic end extending as a spike away from the viral surface (Palese and Shaw, 2007). The monomeric HA molecule is initially synthesized as a single polypeptide chain (HA0) which undergoes posttranslational editing and modification, such as signal peptide cleavage, glycosylation, palmitoylation, and cleavage of the HA0 precursor (Klenk et al., 1975; Lazarowitz and Choppin, 1975; Porter et al., 1979). The resulting HA1 and HA2 subunits from HA0 cleavage are covalently linked by a single disulfide bond, while three HA1-HA2 monomers are associated via non-covalent forces. The HA molecule is structurally composed of two domains, a globular head and a fibrous stem (Stevens et al., 2004; Wang and Palese, 2011). The membrane-distal globular head region is exclusively composed of HA1 subunit and frames the receptor-binding site (RBS) (FIG. 3). The stem region, which is more proximal to the viral membrane, consists of residues from both HA1 and HA2 subunits. Receptor binding and membrane fusion activities are the two major functions of HA during the IAV life cycle, but it has been also suggested that HA may be involved in the virion assembly and budding steps. Initially, HA binds to the terminal sialic acid of oligosaccharides on glycoproteins or glycolipids of the host cells via a shallow depression at the globular head of HA1 subunit (Skehel and Wiley, 2000). After sialic acid receptor binding and endocytosis, the low pH in the endosomes triggers a dramatic conformational change of the HA2 subunit to execute the fusion activity (Stegmann, 2000). When it comes to the egress of progeny viruses, the HA is believed to incorporate the apical transport signals within its transmembrane domain and/or cytoplasmic tail (Brewer and Roth, 1991; Lin et al., 1998). The highly conserved transmembrane domain as well as the cytoplasmic tail of HA has also been shown to be important for efficient raft association which is believed to provide a platform for virion assembly and budding (Melkonian et al., 1999; Takeda et al., 2003).
The major protective immunity (i.e., neutralizing antibodies) against influenza viruses is induced by the HA glycoprotein. As mentioned above, the antigenicity of influenza A virus HA is subtype-specific and this feature is largely determined by the immune response to the globular head region (HA1 subunit). It is generally appreciated that influenza A virus HA possesses five different antigenic sites in the three-dimensional structure, although some overlap of these domains has been noted (Wiley and Wilson, 1981; Caton et al., 1982). For the H3 subtype, the five antigenic sites are A, B, C, D, and E (Wiley and Wilson, 1981), and for the H1 virus strains they are designated as Ca1, Ca2, Cb, Sa, and Sb (Caton et al., 1982). All these sites were identified from amino acid variations in escaping mutants selected with monoclonal antibodies and natural variants as well. These sites are also recognized as neutralizing epitopes, meaning monoclonal antibodies to each one of the five sites can neutralize the infectivity of the virus (Wiley and Skehel, 1987; Wiley and Wilson, 1981). It is notable that the gain or loss of N-linked glycosylation sites in the HA can also significantly affects the antigenicity of the molecule (Caton et al., 1982; Skehel et al., 1984).
NA is a type II transmembrane glycoprotein anchored into the viral membrane by the uncleaved signal/anchor domain at its N-terminus with a six amino-acid tail (Colman, 1994). The monomers of NA are synthesized in the ER and rapidly form a disulfide-linked dimer, which are then non-covalently oligomerized into tetramer. During the transport through Golgi apparatus, NA acquires a complex carbohydrate modification (Hogue and Nayak, 1992). The mature NA glycoprotein homotetramer is composed of a mushroom-shaped head and a slender stalk. The mushroom-shaped head is arranged with four identical coplanar and roughly spherical subunits in circular 4-fold symmetry and stabilized by calcium (Colman et al., 1983; Russell et al., 2006). Each monomeric subunit displays a symmetrical structure of six topologically identical anti-parallel β-sheets arranged as a propeller blade with the enzyme active site located in the center (Colman et al., 1983; Russell et al., 2006). The enzymatic activity on the surface of influenza viruses was first described by George Hirst (Hirst, 1942), who discovered that red blood cells turned out to be refractory to re-hemagglutination by influenza viruses after pre-treatment with the viruses. NA plays at least two major roles in the viral life cycle on the basis of its receptor-destroying activity. First, since the respiratory mucosal layer is also rich in sialic acid receptors for HA, the NA is needed to minimize the binding of HA to “decoy” receptors, thereby enabling the viruses to reach target cells on the respiratory epithelium (Palese et al., 1974). Another well-documented function of NA is to facilitate release of progeny virions by removing the sialic acid receptors from the viral particles and the infected cell surface. This role of NA was interpreted from the finding that intact virions were shown to aggregate on the surface of cells infected by mutants with defects in NA (Palese et al., 1974; Palese and Compans, 1976). Similar to HA, NA has also been reported to play a role in raft association and virion assembly. NA contains signals for apical transport in its transmembrane and cytoplasmic domains. The transmembrane domain also contains a signal for raft association which is separated from the apical sorting signal (Barman and Nayak, 2000). The highly conserved six amino-acid cytoplasmic tail of NA is also involved in virion assembly and budding (Jin et al., 1997).
The IAV NA generally has four antigenic sites consisting of multiple epitopes (Webster et al., 1984). Depending on their relationship with the enzymatic center, antibodies targeting some sites but not all of them can inhibit neuraminidase activity, and the mutations mainly occur in the distal surface loops connecting the various strands of β-sheets (Air et al., 1985). The site-specific mutagenesis indicates that only a few amino acids on five polypeptide loops surrounding the enzyme active site is critical for antibody recognition (Nuss et al., 1993).
Viral Life Cycle
IAV initiates infection by binding the cellular receptor through the HA glycoprotein. Although neuraminic acid serves as the ubiquitous receptor for IAV, viral Has isolated from different species do have differential binding specificity to the linkage between N-acetylneuraminic acid and the penultimate galactose sugar. Typically, human influenza viruses prefer the α-2,6 linkage whereas avian viruses are most likely to bind sialic acid with an α-2,3 linkage (Connor et al., 1994), however this specificity is not absolutely exclusive. After binding to the receptors, the attached virion undergoes a receptor-mediated endocytosis via different pathways (Lakadamyali et al., 2004). The low pH in the late endosome triggers a conformational change in the cleavage-activated HA to initiate fusion of the viral and vesicular membranes to release the core virion into the cytoplasm (Skehel et al., 1982). Meanwhile, M2 proteins channel protons into the core virion, which is believed to promote dissociation of the M1 from the ribonucleoprotein complexes (RNPs) and allow the RNPs to migrate to the nucleus (Pinto et al., 1992; Zhirnov, 1990). The proteins associated with RNPs including PB2, PB1, PA and NP all possess nuclear localization signals, which mediate the active nuclear import of RNPs via interactions with host nuclear import machinery (Kemler et al., 1994). When the RNPs reach the host cell nucleus, the associated polymerase complexes start primary transcription of mRNA from the viral genome by ‘snatching’ a 5′-capped primer from host cellular mRNA (Krug et al., 1979). The primary transcripts are then used in the production of viral proteins by the cellular translation machinery. The replication of viral RNAs occurs through a two-step primer-independent process. A full-length viral complementary RNA (cRNA) has to be synthesized first to serve as the template for the following production of viral RNAs. It was believed that the synthesis of cRNA was delayed until viral proteins had been produced (Beaton and Krug, 1986). However recent findings indicate that cRNA may start to be synthesized in early infection, but it is degraded rapidly by cellular nucleases until sufficient polymerases and NP proteins encapsidate it (Vreede et al., 2004). Newly synthesized polymerases (PB2, PB1, PA) and NP proteins are transported into the nucleus after translation in the host cell cytosol to assemble new RNP complexes with progeny viral genomic RNAs in the nucleus. After their translation, viral membrane proteins HA, NA, and M2 are translocated into the lumen of the endoplasmic reticulum (ER) where they are further oligomerized, glycosylated and subsequently transported to the plasma membrane (Doms et al., 1993). The apical localization of viral membrane proteins in polarized cells determines the assembly and budding site for the progeny viral particles and the apical sorting signals have been identified within the transmembrane domains (TMD) of HA and NA (Nayak et al., 2004). M1 has been shown to be the only absolutely required viral protein during the virion assembly and budding process because of its interactions with other viral components (Gomez-Puertas et al., 2000). M1 and NEP/NS2 are proposed to cooperate with the cellular export factor (CRM1) to direct the nuclear export of viral RNPs (Neumann et al., 2000). After RNPs leave the nucleus, M1 may function as a molecular ‘glue’ to direct RNPs to the assembly site through its interaction with the cytoplasmic lipid membrane and cytosolic tails of integral viral proteins (Ali et al., 2000; Bucher et al., 1980; Enami and Enami, 1996; Ruigrok et al., 2000). Furthermore, M1 is vital for bud formation since budding cannot occur in the absence of M1, yet M1 alone can induce the formation of virus like particles (Gomez-Puertas et al., 2000). When a progeny viral particle is completely formed, the neuraminidase activity of the NA protein will cleave the sialic acid residues on the cellular receptor and those between the new virions to release the viruses for the next round of infection on neighboring cells (Palese et al., 1974).
Influenza is an acute viral infection caused by influenza viruses. It is one of the most common respiratory infections in humans and perhaps one of the most significant with its existence recorded in human history with high morbidity and mortality rate. Careful retrospective investigations of the historical records have revealed that outbreaks of influenza epidemics or pandemics can be traced back to at least the Middle Ages, if not earlier (Kilbourne, 1987). Seroarcheological studies have also shown that 90% of subjects born between 1857-1877 were found to have antibodies to ‘Hong Kong’ influenza virus (H3N2) prior to its epidemic reappearance in 1968 and 26% had pre-epidemic antibodies to the ‘Asian’ influenza virus (H2N2) that caused the pandemic of 1957 (Davenport, 1977; Masurel and Marine, 1973). Also, serological evidence for circulation of H1N1 viruses before the outbreak of 1918 ‘Spanish’ influenza has been documented as well (Masurel and Heijtink, 1983; Rekart et al., 1982).
Seasonal influenza epidemics occur every year in temperate climates mostly from late autumn throughout the next spring with peak periods lasting 6-8 weeks. The seasonality of influenza activity is less established for tropical and sub-tropical regions. However, influenza viruses in the tropical and subtropical areas can circulate throughout the year at relatively low level with typical peaks of activity occurring in the summer months (Reichelderfer and Kendal, 1989). Although epidemics of influenza occur every year, the rates and severity of illness varies substantially from year to year. During a typical influenza epidemic, the overall infection rate is estimated to be 10-20%, but in selected populations or age groups, e.g., school-age children, a rate of primary influenza illness of 40-50% is not uncommon (Glezen, 1996). It is estimated that every year influenza epidemics result in about three to five million cases of severe illness, and about 250,000-500,000 deaths worldwide (W.H.O., 2009). In the United States, an annual average of more than 200,000 hospitalizations and about 36,000 deaths are caused by influenza-associated respiratory and circulatory illnesses (Thompson et al., 2003; Thompson et al., 2004).
In addition to annual seasonal influenza epidemics, pandemics of influenza have emerged at irregular intervals and varied in severity from mild to catastrophic. During the 20th century, there were at least three indisputable influenza pandemics: 1918 ‘Spanish’ influenza, 1957 ‘Asian’ influenza, and 1968 ‘Hong Kong’ influenza. As the worst influenza pandemic in recorded history, the 1918 ‘Spanish’ flu was estimated to cause approximately 675,000 total deaths in the United States and have killed up to 50 million people worldwide (Johnson and Mueller, 2002). In 1957, the ‘Asian’ influenza pandemic caused a total global excess mortality of over 1 million deaths, while the 1968 ‘Hong Kong’ influenza pandemic also resulted in about 1 million excess deaths worldwide (Lipatov et al., 2004). In 2009, the world encountered the first influenza pandemic of the 21st century, which spread more rapidly across the continents than the previous pandemics, probably due to the sharp increase of individual travel. The illness caused by the 2009 H1N1pdm virus was relatively mild in most cases. According to the estimates by CDC from April 2009 to April 2010, more than 50 million people in the United States were infected by the 2009 H1N1pdm influenza virus, which resulted in about 195,000-403,000 hospitalizations and approximately 12,470 deaths in the United States (CDC, 2010). Influenza is generally accepted as an acute, prostrating, self-limited respiratory illness. The incubation period for influenza is relatively short with a typical 1-2 days from infection to onset of illness. The clinical expression of influenza infection is highly variable and largely influenced by the age, physiological status and pre-existing immunity of the host (Kilbourne, 1987). The typical symptoms of influenza infection in adults include fever, chills, headache, sore throat, dry cough, nasal discharge, myalgia, anorexia and malaise, while gastrointestinal symptoms such as vomiting, abdominal pain and diarrhea are also frequently observed in children. Generally influenza is a short-lived infection in healthy adults as most people recover from fever and other symptoms within a week without requiring medical attention, while cough and malaise may persist for one or more weeks after fever has subsided (Cox et al., 2010). Common complications of influenza infection include secondary bacterial pneumonia and exacerbation of underlying chronic cardiac, pulmonary, or metabolic diseases and otitis media in children (Nicholson, 1992). Secondary bacterial infections usually occur 5-10 days after initial onset of influenza symptoms and are responsible for most pneumonia during influenza epidemics. Typically Streptococcus pneumoniae, Staphylcoccus aureus and Hemophilus influenzae are the most common causative pathogens (Schwarzmann et al., 1971). Other uncommon complications of influenza include myositis, myocarditis and pericarditis, acute renal failure, encephalopathy, encephalitis, transverse myelitis, toxic-shock syndrome and Reye's syndrome. The Reye's syndrome is generally associated with the use of salicylate medications in children with influenza-like illness (Noble. 1982).
Currently there are two measures employed to reduce the impact of influenza: antiviral drugs and vaccination, antivirals and vaccines. Antiviral drugs are utilized as chemotherapy as well as chemoprophylaxis to control influenza. Based on the chemical properties and spectrum of activity against influenza, the currently licensed antiviral drugs can be classified into two categories, adamantine derivatives (amantadine and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir). Both drugs have been shown to be effective in decreasing viral shedding and reducing the duration of symptoms of influenza infection by approximately one day if administered within 48 hours of the onset of illness compared with placebo administration (Burch et al., 2009). Adamantane only inhibits the replication of influenza A viruses, while the NA inhibitors are active against both type A and type B viruses, and the recommended treatment course for both antivirals is usually 5 days. Both adamantane derivatives and neuraminidase inhibitors are effective to be used as chemoprophylaxis. When used for chemoprophylaxis, amantadine and rimantadine are approximately 70-90% effective in preventing illnesses resulting from influenza type A infection (Hayden et al., 1996). Zanamivir and oseltamivir are approved to be used prophylactically for influenza A and B infections in individuals aged more than five years and one year old, respectively. Up to 82% of febrile, laboratory-confirmed influenza illnesses were prevented by zanamivir or oseltamivir prophylaxis (Hayden et al., 1999; Welliver et al., 2001). Studies also show that prophylactic treatment of household members with zanamivir or oseltamivir reduced secondary transmission by 79%-89% (Hayden et al., 2000; Welliver et al., 2001). The antiviral activity of amantadine and rimantadine is believed to be exerted through the M2 ion channel functions. At the early stage of the viral replication cycle, the blockage of the M2 transmembrane domain by the drugs prevents the import of protons into the viral core and in turn inhibits the dissociation of M1 from the ribonucleoprotein complex, a step which is essential for the initiation of viral transcription and replication (Pinto et al., 1992; Sugrue et al., 1990). In addition, for certain avian viruses these compounds can block a low pH-mediated maturation of the HA protein during its transport from ER to the cell surface, and as a result the viral assembly process is disrupted (Takeuchi and Lamb, 1994).
Oseltamivir and zanamivir are analogues of sialic acid that block the enzymatic activity of NA to impair the second round of infection by progeny viruses and consequently provide antiviral activity. Because NA activity is essential for newly assembled virions to be released from infected cells and prevent them from aggregating with each other, an effective level of NA activity is critical for multiple viral infectious cycles to generate a successful influenza infection (Gubareva et al., 2000); inhibition of NA activity would therefore attenuate secondary viral infection. The use of adamantane derivatives has been associated with the rapid selection and development of resistant virus strains. Resistant viruses can emerge when either of these drugs is administered for treatment in adults or children. The acquisition of resistance is not associated with attenuation since resistant mutants are equally pathogenic as their drug-sensitive counterparts (Hayden et al., 1996). The resistant strains are mostly found in the H3N2 subtype isolates rather than H1N1 isolates; in 2006, 92% of H3N2 isolates from the United States were shown to be drug-resistant (Bright et al., 2006). Most H5N1 isolates are resistant to the adamantane drugs, therefore neither amantadine nor rimantadine are recommended for prevention or treatment of the highly pathogenic avian influenza infections (Schünemann et al., 2007). Furthermore, all 2009 H1N1pdm viruses are also resistant to the adamantane derivatives, due to a single amino acid mutation at the transmembrane region of M2 (Dawood et al., 2009). Emergence of NA inhibitor resistant variants can be induced in vitro, but requires multiple passages in cell culture (Gubareva et al., 1997). In contrast to the adamantanes, the frequency of isolating naturally occurring NA inhibitor resistant mutants is relatively low (Gubareva et al., 2000), with most cases from children (Kiso et al., 2004). In 2008 a high percentage of seasonal H1N1 strains were found to be resistant to oseltamivir but still sensitive to zanamivir; however H3N2 and type B viruses are still sensitive to both neuraminidase inhibitors. Oseltamivir-resistant H5N1 viruses have also been reported in both recovered and fatal cases (De Jong et al., 2005). Although 2009 H1N1pdm viruses still remain sensitive to NA inhibitors, some oseltamivir-resistant viruses have been reported (Baz et al., 2009).
With the rapid emergence of antiviral drug-resistant influenza viruses, immunoprophylaxis with seasonal or pandemic vaccines still remains the most effective way to control influenza. Since the first inactivated influenza vaccine was used in the 1940s, the effectiveness of inactivated vaccine has been widely demonstrated in both military and civilian populations (Couch et al., 1986; Monto and Terpenning, 1996). Current inactivated vaccines can protect 70-90% of normal healthy adults against naturally occurring disease when the antigens of the vaccine match the circulating influenza viruses (Buxton Bridges et al., 2000; Nichol et al., 1995). Numerous studies have shown that seasonal vaccination reduces rates of hospitalization and death among nursing home residents whose average age is 85 years old (Ohmit et al., 1999; Patriarca et al., 1985). During the 2012-13 season, an interim estimate of the overall effectiveness of influenza vaccine was 56% (95% confidence interval [CI]=47%-63%)(CDC, 2013). Generally, vaccination is associated with reductions in: (a) influenza-related respiratory illnesses and physician visits; (b) hospitalizations and deaths among people at high risk; (c) Otitis media among children; and (d) work absenteeism levels in adults (WHO Global Influenza Surveillance Network, 2011).
Current seasonal influenza vaccines have a trivalent formulation which contains antigens of two influenza A subtypes (H1N1pdm and H3N2) and one or two representative type B strains. Since influenza viruses are highly mutable resulting in antigenic drift, the formulation of influenza vaccine needs to be updated annually. Typically, one or two components of the vaccine will be changed each year. Every February and September the World Health Organization (WHO) makes recommendations for northern and southern hemisphere vaccine formulations, respectively, about the influenza strains that should be included into the vaccine for the following influenza season (Gerdil, 2003). Such a recommendation is based on data collected within the WHO global influenza surveillance network to match the antigenicity of the influenza viruses that are likely emerging and circulating in the following influenza season (Cox et al., 2010). The inactivated influenza vaccines, i.e., flu shot, comprise the vast majority of vaccine doses distributed during annual vaccination campaigns. Since the 1970-1971 influenza season, the vaccine seeds used for production of type A components of the vaccine are high-yield reassortant (HYR) viruses instead of wild type (WT) field isolates (Kilbourne, 1969). By incorporating the “internal” genes from an egg-adapted laboratory strain, A/Puerto Rico/8/1934 (PR8), the reassortants achieve high-yield in eggs while preserving the antigenicity (HA and NA) of the target WT viruses.
Two types of influenza vaccines that are currently licensed in the United States are live attenuated influenza vaccine (LAIV) and inactivated influenza vaccine. The LAIV is composed of viruses that are avirulent and only produce mild or no symptoms on infections. The temperature-sensitive property of LAIV strains limits the replication in human lower respiratory airway since they are cold-adapted with efficient replication at 25 (Smith et al., 2006). In the United States, LAIV is currently only approved for use in healthy individuals aged 2-49 years who are not pregnant (Harper et al., 2004). The inactivated influenza vaccine can be further categorized into two types based on their effective components. The inactivated subvirion vaccine contains detergent-disrupted inactivated virus while surface antigen vaccine only contains isolated hemagglutinin (HA) and neuraminidase (NA) proteins, and both inactivated vaccines are approved for use in people aged more than 6 months old (Harper et al., 2004). Trivalent inactivated vaccine has been shown to be more effective than LAIV in the elderly (Treanor and Betts, 1998), whereas the reverse is true in young children (Belshe et al., 2007). The inactivated influenza vaccines comprise the majority of vaccine doses used during the annual vaccination campaign. Briefly, vaccine strains are grown individually in the allantoic cavity of embryonated chicken eggs, the allantoic fluid is harvested and then the virus is purified and concentrated by zonal centrifugation or column chromatography and finally inactivated with formalin or beta-propriolactone (Gerdil, 2003). For the current influenza vaccine only HA antigens are quantified by the single radial immuno-diffusion assay using standard antigens and specific sheep antiserum (Cox et al., 2010). Each vaccine dose must contain 15 μg HA per virus strain, while the quantity of NA may vary between vaccines.
Since the 1970-1971 influenza season, the vaccine seeds used for growth of type A components of the vaccine are high-yield reassortant viruses instead of wild type (WT) field isolates (Kilbourne, 1969). By incorporating the ‘internal’ genes from an egg-adapted laboratory strain, A/Puerto Rico/8/1934 (PR8), the reassortants achieve high-yield/growth in eggs while preserving the antigenicity (HA and NA) of the target WT viruses. Field isolates of representative type B influenza viruses with relatively high growth in eggs have been utilized usually utilized for production of the B component, however recently manufacturers have started to use type B reassortant viruses derived from reassorting HA and NA genes of WT viruses with ‘internal’ genes of a high growth egg-adapted B virus.
Reassortment generally refers to the “shuffling” of genetic material of a species into new combinations in different individuals. In particular, reassortment of influenza viruses is the rearrangement of viral RNA segments into progeny virus when two or more different influenza viruses infect the same cell. During the assembly of the new progeny virions, each of the RNA segments can derive from either parental virus to result in different gene combinations. Those progeny viruses with mixed-origin RNA segments are called ‘reassortants’. Reassortment occurs in nature within the same type of influenza A, B and C viruses, but not across the different types (Wright, Neumann, Kawaoka, 2007a). The three latest influenza pandemics were all caused by reassortment within IAV. The ‘Asian’ influenza in 1957 and the ‘Hong Kong’ influenza in 1968 were caused by reassortment between human and avian viruses (Kawaoka et al., 1989), while the 2009 pandemic influenza strain was a “triple” reassortant of human, avian, and swine influenza viruses (Trifonov et al., 2009). Although reassortment has evolutionary benefits for the virus, it also provides us with a way to defend ourselves from influenza. In the 1960s, Dr. Edwin D. Kilbourne utilized reassortment as a genetic manipulation tool to quickly introduce desirable properties for vaccine production from a high-yield laboratory strain into a low-yield wild type virus (Kilbourne and Murphy, 1960). Since the application of reassortants to influenza vaccine production in 1971, reassortants have greatly improved the mass production of influenza vaccine. In brief, the seed viruses for the influenza A components are high-yield (hy) reassortants generated in embryonated chicken eggs (in ovo), which must contain two genes for the surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) from the currently circulating WT virus and up to six genes for the ‘internal’ proteins from the hy-donor virus, A/Puerto Rico/8/1934 (PR8) as the backbone (FIG. 1).
For the present classical reassortment method to generate seed viruses, the correct progeny viruses with WT HA and NA are selected for growth by using polyclonal antibodies (pAbs) against the high yield (hy) donor virus. Due to the uncertain effectiveness of pAbs, this negative selection process usually is repeated in 3 cycles and takes about 3-4 weeks or more. As a result, only the viruses with correct matching antigenicity (i.e., WT HA and NA genes) will comprise the selected virus population. With amplification, positive selection occurs with the reassortant variant with the best growth capability out-growing other reassortants. With cloning by limiting dilution the most abundant virus (indicating the virus with the best growth ability) will be readily cloned out from the virus population. The identity of the final cloned virus will be evaluated through molecular and serological tests.
Up to now the vaccine seed viruses prepared via the classical reassortment method are still being predominantly used for influenza A vaccine production because of their superior in ovo growth properties compared to seed viruses made by reverse genetics. Since classical reassortment develops seed viruses entirely in embryonated chicken eggs which are the predominant platform for producing influenza vaccines, the classically-made seed viruses are better adapted to in ovo growth than seed viruses prepared by other approaches.
Besides the established classical reassortment method, the reverse genetics (RG) method is widely recognized and employed as an alternative for developing influenza A vaccine seed viruses.
The RG technique provides an alternative way to make suitable vaccine seed viruses for the large scale production through direct molecular manipulations. The RG system was initiated when Palese and colleagues established a system that incorporates artificial viral RNA (vRNA) derived from cloned DNA into an influenza virus (Luytjes et at, 1989). However, the application of this system is limited by the reconstitution of the RNP complex and the selection of recombinant influenza virus from the helper virus background (Neumann and Kawaoka, 2002). The RG system, which only relies on DNA plasmids to generate vRNA and mRNA for the de novo synthesis of influenza virus, was independently developed by Neumann et al. and Fodor, et al. in 1999 (Fodor et al., 1999; Neumann et al., 1999). This system utilizes RNA polymerase I-driven plasmids to produce the complete set of eight influenza vRNA segments, and RNA polymerase II-driven plasmids for the generation of the three viral polymerases and NP proteins. Therefore, to successfully rescue a live influenza virus entirely from DNA plasmids, a total of 12 plasmids have to be simultaneously transfected into the same eukaryotic cell (e.g. 293T cells). The RG system was later refined by Hoffmann et al. (Hoffmann et al., 2000) to combine the vRNA and mRNA synthesis on one bi-directional plasmid which reduces the total number of plasmids that are required for rescue to eight. In 2005, Neumann and co-workers (Neumann et al., 2005) further reduced the total number of plasmids by combining eight RNA polymerase I transcription cassettes into one plasmid and the RNA polymerase II transcription cassettes of the three viral polymerases into another single plasmid. Several influenza inactivated vaccine seed viruses including highly pathogenic avian influenza virus, H5N1, have been generated by employing RG either through the 12 or eight plasmids system (Hoffmann et al., 2002; Nicolson et al., 2005; Webby et al., 2004). Following the same principle as classical reassortment, to acquire avirulent and high yield properties desired by vaccine manufactures, the HA and NA genes derived from circulating WT viruses are incorporated into a ‘backbone’ containing the six ‘internal’ genes from the by donor, PR8. The biggest advantage of RG over the classical reassortment method in making vaccine seed viruses is the capability of direct genetic modification of the viral genes. Because of the high virulence of the HA of highly pathogenic avian influenza virus (HPAI), the poly basic amino acid stretch at the HA cleavage site has to be removed to make vaccine seed virus attenuated for virus propagation in embryonated chicken eggs. This could only be achieved by RG but not by classical reassortment since the unmodified HPAI will kill the chicken embryo in the embryonated eggs. In addition, the RG technique does not need any selection process which theoretically should prepare the vaccine seed viruses more promptly than the classical reassortment method. However, during the response to 2009 H1N1pdm influenza pandemic the very first vaccine seed candidate was developed by classical reassortment instead of the RG method (Wanitchang et al., 2010). This suggests that although RG represents modern and advanced technology, the classical reassortment still remains as a standard and reliable method for generating seed viruses for influenza vaccine production. Therefore, improving the classical reassortment method will be of great interest to public health preparation against both annual influenza epidemics and potential pandemics.
Other than a few published articles (Kilbourne et al., 2004; Webster et al., 1988), the role of NA mAbs in countering influenza viruses remains to be further elucidated. In the aforementioned publications, NA mAbs were found to inhibit virus release from host cells resulting in size reduction of plaques, and some of these mAbs which inhibited catalytic activity of NA could neutralize the virus in embryonated chicken eggs. These studies merely postulated that mAbs to some epitopes on the NA protein may inhibit virus release more efficiently than others, depending on their relation to the enzymatic center (Webster et al., 1984). Subsequently, in the late 1990s, some mAbs prepared against the NA of A/Beijing/32/92 were shown to provide NA inhibition and also neutralize virus in infected cells (Aymard et al., 1998). In addition, in vivo protection of NA mAbs was demonstrated by treating influenza virus-infected SCID mice with non-neutralizing NA mAb that resulted in reduced pulmonary virus titer load (Mozdzanowska et al., 1999). However, there are few documented use of monoclonal antibodies in screening of seed viruses, particularly, seed viruses which can propagate and serve as immune compositions or vaccines for the prevention and treatment of influenza.
In this sense, polyclonal antibodies (pAbs), which have been traditionally employed in classical reassortment methods, are laden with problems. Firstly, classical methods involving pAbs are time-consuming and irreproducible due to the intrinsic disadvantage of using variably effective pAb. Additionally, polycloncal antibodies are problematic due to uncertainties of specificity and/or cross-reactivity. Due to time-sensitive nature of vaccine development and deployment, pAbs are not very useful as the selection by pAbs has to be performed three times or more to guarantee the elimination of donor virus' HA and NA genes (i.e., to ensure that the screened candidates only incorporate HA and NA genes from wild type (WT) viruses). This inherent disadvantage has led to increased interest in reverse genetics (RG) technique, which does not require such a selection process for vaccine seed virus preparation; the RG technique can directly manipulate and fix the gene composition for the resultant vaccine seed viruses. However, to date, HYRs prepared by classical reassortment have better growth properties than HYRs prepared by RG.
Monoclonal antibodies, which can be generated to meet predefined specificity requirements (see Kohler and Milstein, 1975), have been used in other research applications, e.g., immunostaining and immunoblotting; however, their application in the production of seed viruses has been discouraged in the art. For example, there are scientific reports of HA mAbs rendering viruses non-infective both in vitro and in vivo. There are only a few reports of non-neutralizing HA mAbs in literature (Cascino et al., 1986; Vanlandschoot et al., 1998); however, their use in screening of seed viruses, particularly, with respect to epitope mapping, was previously unknown in the field. Thus, there is an unmet need for novel antibody compositions which can be employed more efficiently to screen candidate viruses that serve as (or provide) immunotherapeutic compositions and vaccines, in particular monoclonal antibodies which can be used as reagents in classical reassortment for the generation of seed viruses, including seed viruses to fulfil the growing need for prophylactic or therapeutic vaccines against the influenza viruses, as well as serve as diagnostic and screening tools for the identification of new strains of viruses in circulation.